TSA (thiol-specific antioxidant) is a protein that specifically protects enzymes from oxidative inactivation by thiol metal-catalyzed oxidation systems. The most studied antioxidant property of TSA is the protection of glutamine synthetase from oxidative damage by the DTT/Fe(III)/oxygen system. Thiol oxidation was measured quantitatively by a polarographic oxygen consumption technique. TSA inhibits oxygen consumption during DTT oxidation, but not curing ascorbate oxidation in reactions catalyzed by different ferric complexes. Catalase and glutathione peroxidase are also efficient inhibitors of oxygen consumption by DTT. The oxygen consumption results correlate very well with the glutamine synthetase inactivation experiments. The kinetics of oxygen consumption during DTT oxidation contains two phases. Hydrogen peroxide, which is produced during the lag phase, initiates the propagation step of the kinetics. TSA inhibits efficiently oxygen consumption by DTT only when added prior to the beginning of the kinetics. Addition of TSA at concentrations below 20 mug/ml induced an increase in the lag phase. Catalase provokes similar effects, suggesting that TSA inhibits oxygen consumption by DTT through the disposal of hydrogen peroxide. Exogenous hydrogen peroxide addition eliminates the TSA inhibitory effect on the oxygen consumption by DTT, and also its protection of glutamine synthetase from oxidative inactivation. The hydrogen peroxide-removing activity of TSA was confirmed by studies with the glucose/glucose oxidase and methanol/alcohol oxidase systems. Two sulfhydryl groups are consumed per hydrogen peroxide removed and one disulfide bond is produced, demonstrating that TSA is a thiol peroxidase. This report shows that the thiol peroxidase activity of TSA is involved with its antioxidant property.